The in-hospital mortality was 30/107 (28%). GNB late-onset sepsis (LOS) was associated with an increase in mortality before discharge compared to uninfected controls  but not versus suspected sepsis. The suspected LOS cases did not statistically differ significantly from uninfected controls. The case fatality rate (CFR) at 5 days was 5/33 (15%) in GNB early-onset sepsis (EOS) and 25/74 (34%) in GNB-LOS. The adjusted hazard for 5 days CFR was higher in GNB-LOS versus uninfected controls , but no significant difference was seen in GNB-LOS versus suspected sepsis or in suspected sepsis versus controls. ESBL production was seen in 7/107 (6.5%) of the GNB isolates. GNB-LOS was associated with a higher 5 days CFR and in-hospital mortality compared to uninfected controls but not versus suspect sepsis. The incidence of both GNB-EOS and GNB-LOS was lower than previously reported from comparable high-income settings. The occurrence of antibiotic resistance was low.This study is to determine the incidence and outcome of neonatal gram-negative bacilli (GNB) sepsis in Stockholm, Sweden, and describe bacterial characteristics. This is a retrospective cohort study. All infants with GNB-sepsis between 2006 and 2016 were included and matched with two control groups, with suspected sepsis and uninfected neonates, respectively. Outcome was death before discharge, risk of death within 5 days after sepsis onset, and morbidity. The resistance pattern from all GNB was collected, and all available isolates were subjected to genome typing. All neonates with GNB-sepsis (The online version contains supplementary material available at 10.1007/s10096-021-04211-8. Neonatal infections account for more than one-third 36%) of all neonatal deaths globally. Sepsis is the leading cause of neonatal mortality and accounts for more than one million deaths/year worldwide , 2. In h6% of allThe growing challenge of antimicrobial resistance (AMR) in neonatal intensive care units (NICUs), especially with resistant GNB, is associated with a high mortality and poor long-term outcome , 10–12. Early diagnosis and treatment of neonatal sepsis are difficult, and the fact that a consensus definition of neonatal sepsis is lacking makes it even more challenging –18. The We aimed to analyze the incidence of neonatal GNB-sepsis and associated mortality and morbidity in neonates in our setting. We also wanted to determine whether there were differences in outcome between patients with culture proven sepsis, suspected sepsis (negative blood culture) and uninfected patients. We characterized the invasive bacterial isolates as to clonality and presence of AMR genes.There are six delivery units and four NICUs in the Stockholm region. A total of 29,553 infants were born alive at these delivery units during 2016. The NICUs are Karolinska Danderyd (level 2), Karolinska Solna (level 3), Karolinska Huddinge (level 3), and Södersjukhuset (level 2). From March 2014 to May 2016, a seventh small delivery unit and levels 1–2 neonatal unit, BB Sophia, operated.E.coli. Infection control routines were similar in all included hospitals.The all-cause neonatal mortality before 28 days of life in the Stockholm region was 0.7–1.8/1000 live born (mean 1.4/1000) (2006–2016). The recommended empiric antibiotic therapy for unknown EOS was since 2012 benzylpenicillin/amikacin and for LOS cloxacillin/amikacin or cefotaxime/amikacin . Between> 5 days. Patients with GPB-sepsis were not analyzed. EOS and LOS were defined according to age at onset of sepsis symptoms before or after 72 h of age.A matched cohort study was undertaken where all neonates with GNB-sepsis at Stockholm’s four NICUs between January 2006 and December 2016 were included. We identified all patients with a positive GNB blood culture, at least two clinical signs  and antibiotic therapy for To identify the sepsis cases, we used the ICD-10 codes for GNB-sepsis in the electronic medical record systems Take Care and Clinisoft and merged them with the Swedish Neonatal Quality Register (SNQ). Patient characteristics from the total NICU-stay were collected.The two groups of controls, manually collected from the same registries, were neonates with suspected sepsis and those uninfected during their NICU-stay. We chose controls with the same gestational age (GA) and closest birth date. Suspected sepsis was defined as the ICD-10 code P36.9, clinical symptoms, a negative blood culture, and subsequent antibiotic therapy for at least 5 days. Uninfected infants, alive at 72 h of age, were defined as not fulfilling the ICD-10 criteria for sepsis or suspect sepsis during the NICU-stay. The proportion of the case vs suspected sepsis vs uninfected was planned to be 1:1:3. The number of uninfected neonates in the same gestational ages as the cases during 2006–2016 was insufficient; hence, the proportion of the cases to controls was 1:1:2.6.There was no variability of GA in the matching groups, but there was variability in closest birth date in the controls depending on GA. The variability in closest birth date between cases in the three matching groups was 10 years, but on average below 24 months.The primary outcome was death before discharge from NICU. The secondary outcomes were sepsis mortality 5 days after onset of GNB-LOS and major morbidities, such as retinopathy of prematurity (ROP), intraventricular hemorrhage (IVH), and bronchopulmonary dysplasia (BPD).Death 5 days following a positive blood culture is presented in the study as 5 days case fatality rate (CFR). The suspected sepsis CFR was death 5 days after onset of therapy for suspected sepsis. Repeated episodes of sepsis were documented in a few numbers of patients, but survival was analyzed from the first invasive GNB episode. Proportions of multidrug-resistant GNB strains and the burden of AMR in clinical samples were determined.Escherichia coli, Klebsiella pneumoniae, K. oxytoca, K. aerogenes, Enterobacter cloacae, Citrobacter koseri, Serratia marcescens, Proteus mirabilis, Pseudomonas aeruginosa, Acinetobacter baumannii, and Haemophilus influenzae. The Neisseria species were considered to be contaminants. Blood cultures with contaminants were not included. All isolates were susceptibility tested for the following: gentamicin, amikacin, trimethoprim-sulfamethoxazole, cefotaxime, ceftazidime, ciprofloxacin, imipenem, meropenem, ertapenem, and piperacillin-tazobactam.GNB in the study refers to the following species: www.eucast.org). MDR was defined as resistance to at least one antibiotic agent in three or more antibiotic groups [All GNB isolates were cultured, isolated, and identified according to routine validated clinical methods and guidelines used during the study period. Antibiotic susceptibility testing was performed by the disk diffusion method and interpreted according to the guidelines of the Swedish Reference Group of Antibiotics before 2011 and between 2011 and 2016 according to guidelines from the European Committee on Antimicrobial Susceptibility Testing  analysis in CLC Workbench [Due to the retrospective design of the study, only 33/107 isolates were available for the genetic analyzes. Whole genome sequencing (WGS) was performed at the Science for Life Laboratory . Multi-locus sequencing (MLST) was performed in silico as described previously . All Entorkbench .t-test and summarized using means and SDs if unimodal, symmetrically distributed variables. If the distribution was skewed, they were shown with median values and ranges. Pearson’s Chi-squared test was used to compare categorical variables. Statistical significance was defined as p values < 0.05, and confidence intervals of 95% were used.This was an open cohort design with varying lengths of time from onset of sepsis to discharge. Comparisons of continuous variables were made with Wilcoxon rank-sum or two sample We used logistic regression to measure odds ratios (OR) of dying, separately for EOS and LOS, and adjusted for different variables in the regression model of EOS and LOS. Variables adjusted for in the EOS group were as follows: gestational age, gender, perinatal antibiotics, birth mode, and prenatal steroid treatment. In the LOS group, we adjusted for gestational age, gender, prenatal steroid treatment, mechanical ventilation, and necrotizing enterocolitis (NEC). We chose these variables since mechanical ventilation is associated with a higher mortality and prenatal steroids with a lower mortality generally. We also adjusted for NEC since we considered NEC to be a confounder in the association between GNB-sepsis and death. Because of the strong correlation between birth weight (BW) and GA, the risk factor BW was excluded from the analysis. In the logistic regression for morbidities , we used composite binary variables for death and the specific morbidity.We analyzed EOS and LOS separately in the survival analysis. The Kaplan-Meier method was used to visualize survival over time. In the survival analysis for 5-day mortality after index day (GNB-sepsis date of the respective case), Cox proportional hazard regression was performed to measure the hazard ratio (HR) for dying between the cases and their matched controls. The HR gives the time-dependent instantaneous rate ratio of dying, but is in this study interpreted as a ratio of risks of death occurring within 5 days, similar to the interpretation of ORs in logistic regression.Covariates adjusted for in the Cox-regression model in the EOS and LOS cohort were the same as in the logistic regression model. Stata Statistical Software version 16.0, StataCorp, TX, USA, and JMP 15.1.0. SAS Institute Inc., Cary, USA, were used.n = 10,418), Karolinska Solna (n = 5828), Karolinska Huddinge (n = 6904), and Södersjukhuset (n = 8728). These four units are levels 2–3 NICUs with a total of 75–80in-patient cots.During the study period, 310,091 infants were born alive at the included delivery units. Of these, 31,878 (10.2%) neonates were admitted to the neonatal units of Karolinska Danderyd , with a cumulative incidence of 0.35 cases per 1000 live born during the study period. Among the infants admitted to the neonatal unit, 1026/31,878 (3.2%) had suspected but not culture-verified sepsis with a cumulative incidence of 3.3/1,000 live born.n = 111), medical records were retrievable in 107 patients, of which 33 were GNB-EOS and 74 were GNB-LOS. These cases were matched with 107 patients with suspected sepsis (culture-negative) and 295 uninfected controls. In total, data from 509 patients were analyzed. The clinical characteristics of included patients are presented in Table Among neonates with invasive GNB-sepsis n = 11, medicMore than one LOS episode was seen in 35/107 (33%) cases where the causative pathogens were GNB and GPB, and 57% (20/35) of them had a GPB-sepsis episode before a GNB-sepsis. The 33 GNB-EOS cases were distributed as 4, 4, 1, 3, 2, 4, 4, 5, 1, 2, and 3 per year during the years 2006–2016. There was no statistical difference in the trend of EOS cases per year during the study period. The 74 GNB-LOS cases were distributed as 4, 9, 12, 5, 11, 8, 5, 3, 4, 5, and 8 which indicate a slight but not statistically significant decrease over the period.p = 0.023), 19 days for GNB-LOS, and 9 for suspected LOS (p < 0.001).The pairwise analysis between groups showed that the median age at diagnosis was 1 day for GNB-EOS, 0 for suspected EOS (p = 0.11), but the GNB-EOS group had a significantly higher use compared to the uninfected group (52% vs 33 %). Similar results were found in GNB-LOS (49%) where use of antenatal antibiotics differed from their uninfected control group (40%) (p < 0.01).’The administration of prenatal steroids did not differ between culture proven GNB-EOS and suspected sepsis. The GNB-EOS group did not differ from the suspected EOS group regarding administration of antibiotics to mothers prenatally  compared to suspected LOS (15%) and uninfected controls (22%)  and uninfected cases . The days of total parental nutrition (TPN) in the GNB-EOS  days were higher and differed significantly from the uninfected group .GNB-LOS and suspected LOS had significantly more days of ventilatory support, umbilical artery catheter (UAC), peripheral central venous catheter (pCVC), and TPN than the uninfected group. Days of TPN and total days with pCVC were significantly higher in the GNB-LOS group compared to the suspected sepsis and the uninfected group Table .Thirty 30/107) neonates with GNB-sepsis died before discharge (5/33 EOS and 25/74 LOS), with a case fatality rate of 28%. The median age at death was 28 days (IQR 14–52) among the infants with GNB-LOS that died during hospital stay. The mortality in the EOS group was too small to make univariate comparisons between the groups relevant. Comparing GNB-LOS with the suspected sepsis and uninfected control groups, the proportion of deaths before discharge was 33.7% (25/74), 18.9% (14/74), and 7.6% (15/196), respectively. The CFR of GNB-LOS in different gestational ages were in GA ≤ 28 , GA 29–32 , GA 33–36 , and GA ≥ 37 . Proportions of deaths of GNB-EOS and GNB-LOS in different gestational ages are presented in Online Resource 07 neonatIn the logistic regression of the relation between GNB-LOS and death, there was a 2.2 times higher odds (crude OR) of dying before discharge at NICU in the GNB-sepsis group (EOS and LOS combined) compared to the suspected sepsis group and 4.8 times higher odds compared to uninfected cases. There was no statistically significant difference in mortality before discharge between patients with GNB-EOS, suspected EOS, and controls. Gestational age was the only factor associated with death in GNB-EOS(Table Neonates with GNB-LOS were 6.5 (crude OR) and 3.9 (CI: 1.6–9.4) (adjusted OR) more likely to die during hospital stay compared to the uninfected matched control group. A higher gestational age was protective. The comparison between GNB-LOS and suspected LOS showed no significant difference in the odds of dying before discharge  (Table p < 0.001) and between GNB-LOS and the suspected sepsis group (p = 0.039) but not between the suspected sepsis group and uninfected controls (p = 0.37). In a Cox-regression model, the adjusted hazard ratio (HR) of dying 5 days after GNB-LOS onset vs uninfected controls was 3.7 (CI: 1.2–11.2), but no increased hazard was seen in GNB-LOS versus suspected LOS (Table The 5 days CFR was 15% 5/33) in GNB-EOS. All neonates with GNB-EOS that died died before 5 days after GNB-EOS onset. The 5 days CFR of GNB-LOS was 17.6% (13/74). The crude 5 days CFR differed significantly between GNB-LOS and the uninfected controls . No difference was seen regarding BPD. Verified GNB-LOS differed from uninfected controls regarding ROP 3–4 , but not regarding IVH 3–4 and BPD  and ROP 3–4  compared to uninfected controls, but no difference was seen compared to suspect LOS.Enterobacterales, comprising E. coli, K. pneumoniae, Enterobacter spp., and S. marcescens. Three other gram-negative species were represented: A. baumannii, P. aeruginosa, and H. influenzae. Proportions of deaths from GNB-EOS and GNB-LOS and the causing pathogen can be seen in Online Resource E. coli and 2/20K. pneumoniae and 2/14E. cloacae. The antibiotic resistance pattern of all isolates is presented in Table All 107 GNB from confirmed positive blood cultures are presented in Online Resource Gram-negative sepsis is an uncommon but serious disorder in the neonate, especially in the premature born –6, 8, 32E.coli-LOS per 1000 NICU admissions was about half that reported in studies from other high-income countries [The incidence of neonatal GNB-sepsis in the region was 0.35/1000 live born neonates and remained unchanged during the study period. The incidence of GNB-EOS was 0.11/1000 live births which is about half of what recently has been reported from the western part of Sweden, where the incidence of GNB-EOS was 0.25/1000 live births . The difountries .We found that the need for intensive care interventions differed between the groups. The GNB-LOS group had significantly more days of supportive intensive care compared to uninfected controls, but not to suspected-LOS. These invasive measures could be risk factors for LOS but also the consequences of infection. As well neonates with suspected sepsis needed more intensive care in terms of mechanical ventilation, parenteral nutrition, and central catheters, than the uninfected controls.The antenatal factors delivery by caesarean section and exposure to prenatal antibiotics occurred more frequently in infants with GNB-LOS than in infants with suspected sepsis or in uninfected controls. A dysbiotic neonatal intestinal microbiota due to C-sectionand/or use of antibiotics has previously been associated as a risk factor for neonatal LOS. The suggested biological rationale is that an altered first-colonizing microbiota cannot confer protection against bacterial translocation in the neonatal intestine , 34.We found GNB-sepsis to be a great risk factor for mortality and show the in-hospital mortality rate to be 28% of all GNB-sepsis cases. The in-hospital mortality rate was more than 2.3 times higher among the infants with GNB-LOS compared to those with GNB-EOS, which possibly reflects the fact that the LOS group were more premature, had lower BW, had more co-morbidities and a longer duration of hospital care. Prenatal steroids have been shown to be protective against a number of morbidities in preterm infants , and in When adjusted for confounders, the GNB-LOS group’s in-hospital mortality was 3.9 times higher compared to uninfected controls. We found no statistical differences in in-hospital mortality between the other control groups.Many studies on neonatal sepsis present crude mortality after a positive blood culture. However, autopsy completion is infrequently performed. We have tried to relate the sepsis episode with sepsis-related mortality and calculated the 5 days CFR. The 5 days CFR for GNB-EOS was 15% and for GNB-LOS 17%. GNB-LOS was most common (70%) in the lower GA (≤ 28 weeks), and the CFR was as high as 35% in this group.From the survival analysis, we concluded that the adjusted hazard for dying within 5 days from the GNB-LOS onset was four times greater than if the neonate was uninfected. There was no statistical significance in the adjusted Cox-regression analysis in comparing the other groups with each other, which possibly might reflect a type II error and the small number of observations. The Kaplan-Meier curve gives us the indication that suspected-LOS is associated with a greater hazard of surviving than in uninfected, but we could not show that statistically. Causal data on reasons for death in the suspected sepsis and uninfected group were not analyzed but could be explained by the most common non-infectious causes of death in the NICU such as respiratory failure, asphyxia, IVH, metabolic disease, and lethal genetic syndromes.Without doubt, culture proven GNB-LOS is related to an increased risk of mortality and morbidity, as previously reported , 8, 32. We could not draw conclusions about the association between GNB-sepsis and severe complications of preterm birth such as BPD and ROP 3–4, as the most severely ill patients died before they could be validated for these conditions. IVH occurs early during the same time frame as GNB-EOS and was also overrepresented in GNB-EOS compared to neonates with suspected sepsis and the uninfected. As IVH often occurs before the onset of GNB-LOS, we did not analyze it in this context. Both ROP 3–4 and BPD was associated with GNB-LOS.E.coli was the most common pathogen causing GNB-LOS with a 5 days CFR of 9%. The highest 5 days CFR (33%) was caused by the Enterobacter spp. We could not statistically relate specific pathogens to mortality which is an important issue for the clinician.E.coli was seen in a large cohort (n = 733) of neonatal E-coli sepsis [Enterobacterales in our study, with a smaller sample size, was 7/107 (6.5%) and is still considered low. The low incidence of GNB-LOS and AMR could be the result of long-standing efforts in infection control and antimicrobial stewardship [E.coli in the region. After that, we could not see any high rates resistance to amikacin or third generation cephalosporins that would lead to any change in the empiric therapy.The rate of antibiotic resistant bacteria in our study was low compared to studies from other settings , 37. In i sepsis . The prowardship , 40. In One strength of the study is its population-based approach as it covers almost all 310,091 infants born in the Stockholm region during the study period. Another strength of the study is that all medical records from the patients with GNB-sepsis, suspected sepsis, and controls were validated against medical records. All data was validated against medical records because between 2006 and 2010, there were no predefined sepsis criteria and data completeness in SNQ regarding causative agents was low in the study region. Sepsis criteria have in later years been standardized, and reporting to SNQ has been changed from retrospective summaries to web-based uploads on a daily basis. In later years, SNQ has been shown to exhibit similar or higher completeness for neonatal sepsis as the Swedish Medical Birth register which is considered to be very high  .The limitations of the study are related to the retrospective design and, despite covering all cases during an 11-year period in an area with more than 2 million inhabitants, the small sample size. The procedure of matching the controls to each sepsis case has been done as accurately as possible. The physiological vulnerability of the neonate in different gestational ages is the most important variable for matching. The size of the cohort makes it impossible to match for more morbidities and is therefore a limitation of the study.We conclude that GNB-sepsis is rare but it remains a serious threat to neonatal patients in the region. GNB-sepsis is a risk factor for neonatal mortality compared to suspect sepsis and uninfected controls. We found a lower incidence of GNB-EOS than previously described in Sweden and other high-income settings, and for the first time, we present the incidence of GNB-LOS in Sweden. The GNB-EOS or GNB-LOS incidence did not change during the study period. The incidence of AMR was low, the AMR pattern did not reveal any highly resistant strains, and the incidence did not change over time. This is reassuring as the current empiric therapy against bacterial sepsis of unknown origin appears to be relevant despite its use over a long period of time.ESM 1(DOCX 51.1 kb)ESM 2(DOCX 258 kb)ESM 3(DOCX 51.3 kb)ESM 4(DOCX 27.1 kb)


Pyricularia oryzae Cav. in Arabidopsis thaliana (L.) Heynh. (Arabidopsis), in diurnal conditions, varies with the inoculation time. Moreover, we indicated that the circadian clock plays an important role in regulating time-of-day differences in NHR to P. oryzae in Arabidopsis. However, the involvement of photoperiod in regulating NHR was still not understood. To determine the photoperiod role, we performed the experiments in continuous light and darkness during the early Arabidopsis–P. oryzae interaction. We found that the light period after the inoculation in the evening enhanced the resistance to penetration. However, the dark period after the inoculation in the morning suppressed the penetration resistance. Furthermore, the genetic analysis indicated that jasmonic acid, reactive oxygen species, and tryptophan-derived metabolite(s) contribute to the photoperiod regulation of NHR in Arabidopsis. The present results denote that photoperiod plays an important role in regulating time-of-day differences in NHR to P. oryzae in Arabidopsis.In plant–pathogen interactions, a proper light environment affects the establishment of defense responses in plants. In our previous experiments, we found that nonhost resistance (NHR) to  Light directly regulates plant physiology and development as well as affects many plant responses to the environment, including defense responses in plant–pathogen interactions ,2,3. LigArabidopsis thaliana (L.) Heynh. (Arabidopsis) mutants with compromised NHR to non-adapted powdery mildew Blumeria graminis f. sp. hordei (Bgh) have been identified: penetration 1 (pen1), pen2, and pen3 ,36pen2-100 a.m.) B with th00 a.m.) B. Col-0 ondition A. In conondition A.P. oryzae, we examined the spore germination rate and the appressorium formation rate at 72 hpi on Col-0 and pen2-1 plants’ leaves. To quantify the rates, we examined the fungal sporelings on six leaves from six independent plants per experiment and genotype . The spore germination rate on Col-0 plants in the PL condition was significantly lower than the rate on Col-0 plants in the PM condition  plants. In these mutant plants, the penetration rates were significantly higher than those of pen2-1 plants in the PM condition, suggesting the severely compromised NHR in these plants. Nonetheless, the penetration rates under the PL condition were significantly lower than those of the PM condition  Jasmonic acid (JA)-related and other defense-related genes ; (2) Reactive oxygen species (ROS)-related genes (AtRbohD and AtRbohF); and (3) Tryptophan (Trp)-derived metabolites-related genes .To identify genes responsible for controlling photoperiod-dependent NHR to  mutants , which wP. oryzae in leaves of all mutants between AM and AD conditions. In these mutants, except for pen2-1 pad3 mutant, the penetration rates of P. oryzae in leaves in the AD condition were significantly higher than those in the AM condition  and restriction of pathogen growth [PR-1 expression [We previously found that NHR to bidopsis ,36. In tractions ,2. As ann growth . FurtherP. oryzae after the morning and the evening inoculation and discovered that the inoculation time had little effect on the appressorium formation rates of P. oryzae [P. oryzae in the PL condition was significantly reduced compared with the PM condition  of PEN3 should not be exported in pen3 plants and that the failure would have a suppressive effect on NHR in the AD condition. As PEN3 functions redundantly with PLEIOTROPIC DRUG RESISTANCE (PDR) 12 in the export of camalexin in Arabidopsis [P. oryzae into leaves of pen2-1 pad3 plants was not different from that of pen2-1 plants under AM and AD conditions  in Arabidopsis.We demonstrated that NHR to P. oryzae in Arabidopsis. Thus, the mechanisms findings can be used to improve plants’ defense responses and contribute to the increased productivity of crop plants in the future.NHR is the most robust and durable resistance in plants. Future studies are needed to understand the genetic and mechanistic mechanisms, including the interaction between the circadian clock and photoperiod of NHR to The plants used in this study were Col-0 (wild-type), pen2-1 , pen2-1 −2sec−1 fluorescent illumination). Then, the plants were transferred to soil and grown in a growth chamber for 4 weeks, where they continued to grow in short-day length conditions (9:15 L:D) at 22 °C (in 100 umol m−2s−1 fluorescent illumination).Arabidopsis plants were grown on Murashige and Skoog plates in a growth room for three weeks in short-day length conditions (9:15 L:D) at 22 °C (in 100 umol mP. oryzae isolate Hoku 1 (race 007) on an oatmeal agar media at 25 °C. Then, we inoculated Arabidopsis with P. oryzae by applying 5 uL droplets (5 × 104 spores/mL) of P. oryzae onto leaves (leaf number 8) of rosettes on Arabidopsis  in the morning and the evening. Then, inoculated plants were maintained in a growth chamber with saturating humidity in short-day conditions (9 h:15 h light:dark) at 22 °C (in 100 umol m−2s−1 fluorescent illumination). Inoculated leaves were harvested at 72 h post-inoculation (hpi).We incubated P. oryzae, we examined the fungal sporelings. This examination was performed on six leaves from six independent plants per experiment and genotype . Experiments were repeated thrice.To quantify the spore germination rates and the appressorium formation rates of P. oryzae by observing autofluorescence or hyphal elongation at infection sites with fluorescence and bright-field microscopy. Each plant genotype was quantified in three independent experiments.To quantify cell penetration, we examined germinated fungal sporelings that had developed appressoria (six leaves from six independent plants per experiment and genotype). We evaluated a minimum of 100 appressoria/leaves. We detected successful penetration of


Maternal hypotension after spinal anaesthesia occurs at a high rate during caesarean delivery and can lead to adverse maternal or foetal outcomes. The aim of this study was to determine the optimal dose of spinal ropivacaine for caesarean section with or without intravenous single bolus of S-ketamine and to observe the rates of hypotension associated with both methods.Eighty women undergoing elective caesarean delivery were randomly allocated into either a ropivacaine only or ropivacaine with intravenous S-ketamine group. If the upper sensory level of the patient reached T6 and the visual analogue scale (VAS) scores remained below 3 points before delivery, the next patient had a 1/9th chance of receiving a lower dose or an 8/9th chance of receiving the same dose as the previous patient. If the patient had VAS scores of more than 2 points or needed an extra epidural rescue bolus before delivery, a higher dose was used for the next patient. The primary outcome was the successful use of spinal ropivacaine to maintain patient VAS score of < 3 points before delivery and the incidence of post-spinal hypotension in both groups. Secondary outcomes included the rates of hypotension-related symptoms and interventions, upper sensory level of anaesthesia, level of sedation, neonatal outcomes, Edinburgh Postnatal Depression Scale scores at admission and discharge, and post-operative analgesic effect. The 90% effective dose (ED90) and 95% confidence interval (95% CI) were estimated by isotonic regression.The estimated ED90 of ropivacaine was 11.8 mg (95% CI: 11.7–12.7) with and 14.7 mg (95% CI: 14.6–16.0) without intravenous S-ketamine, using biased coin up-down sequential dose-finding method. The rates of hypotension and associated symptoms were significantly lower in S-ketamine group than in the ropivacaine only group.A spinal dose of ropivacaine 12 mg with a single intravenous 0.15 mg/kg bolus dose of S-ketamine may significantly reduce the risk of hypotension and induce sedation before delivery. This method may be used with appropriate caution for women undergoing elective caesarean delivery and at a high risk of hypotension or experiencing extreme nervousness.http://www.chictr.org.cn .The online version contains supplementary material available at 10.1186/s12884-021-04229-y. Maternal hypotension is the most common side effect of spinal anaesthesia during caesarean delivery, accounting for 70–83% of all cases –3. It maS-ketamine is an S+ isomer of ketamine that is twice as potent as ketamine ; it achiWe hypothesized that a single ultra-low IV bolus of S-ketamine may reduce the total need for spinal ropivacaine and thus reduce the incidence of post-spinal anaesthesia hypotension during caesarean delivery. Finally, 90% effective doses (ED90) of spinal ropivacaine with and without a single IV bolus of S-ketamine were estimated, using the biased coin up-down (BCUD) sequential dose-finding method; the incidence of post-spinal anaesthesia hypotension was observed.http://www.chictr.org.cn . A total of 85 women, aged 22–40 years, and with a full-term (> 37 weeks of gestation) singleton pregnancy, the American Society of Anaesthesiologists (ASA) physical status II, and undergoing an elective caesarean delivery were recruited during January 2021 and April 2021 at the International Peace Maternity and Child Health Hospital. After providing signed informed consent, the women were allocated by a research assistant to one of two groups, using a random number generator in SPSS for Windows version 18.0 , specifically, ropivacaine alone (R) and ropivacaine with intravenous S-ketamine (RS) groups. All patients were blinded to group allocation.This was a prospective, double-blind, randomised, BCUD-based, sequential dose-finding study approved by China Ethics Committee of Registering Clinical Trials, West Hospital, Sichuan University, Sichuan, China on 29 September 2020  and registered at Exclusion criteria were as follows: contraindications to combined spinal and epidural (CSE) anaesthesia; the ASA score of III–IV; allergy to ketamine, S-ketamine, or ropivacaine; history of gastroesophageal reflux, full stomach, placenta previa, placental abruption, pre-existing or pregnancy-induced hypertension, preeclampsia, cardiovascular or cerebrovascular disease; foetal distress or abnormalities; multiple gestations; refusal to participate, and emergent caesarean delivery. Due to differences in delivery time and uterine contraction pain levels after surgery, women who had previously undergone a caesarean section and multiparas were excluded.− 1 to maintain the vein open.Patients were recruited for the study on the day before surgery in the obstetrical wards, when they were asked to complete the Edinburgh Postnatal Depression Scale (EPDS), which is a screening instrument that helps detect women at risk for depression in the perinatal period . All patRoutine monitoring was performed continuously, including electrocardiography, non-invasive blood pressure, and pulse oximetry. Systolic blood pressure (SBP), heart rate (HR), and pulse oximetry were assessed every 2 min. The first two resting SBP and HR measurements obtained in the supine position were recorded, and their average values were treated as the baseline values. If the baseline SBP was of > 140 mmHg, the patient was excluded from the study due to hypertension.The CSE puncture was performed routinely at the level of L3–4 by an anaesthesiologist with more than 15 years of experience, while the patient was in the right lateral decubitus position. A 17G Tuohy needle was used to perform the epidural puncture with a paramedian approach. After identifying an entrance into the epidural space, a 27G Whitacre needle was inserted through the epidural needle. Once the cerebrospinal fluid was detected, an isobaric ropivacaine dose of 0.75% was injected through the Whitacre needle. After injecting the ropivacaine, an epidural catheter was inserted into the epidural space by advancing it 3 cm through the Tuohy needle. The patient was immediately moved to the supine position with the left uterine displacement created by placing a wedge under the right hip.− 1.h− 1 lactated Ringer’s solution was administered until delivery. A rescue 5-ml bolus of 2% lidocaine was administered through the epidural catheter 2 min before the surgery for patients that could still feel the cold sensation of ice being placed on the skin below the level of T6 and those that complained of pain and whose VAS score was of > 2 points during the surgery. A 5-ml IV dose of 0.15 mg.kg− 1 S-ketamine with normal saline or 5 ml normal saline solution was administered with a 5-ml syringe 1 min before the surgery, depending on group assignment. Hypotension was defined as SBP of < 80% of the baseline value. A rescue dose of 50 μg phenylephrine was administered whenever hypotension was detected. Bradycardia was defined as HR of < 50 beats per minute (bpm) and treated with 0.5 mg atropine. The level of sedation was assessed using a 5-point scale  , SO2, and lactate levels), neonatal weight, Apgar scores measured at 1 min and 5 min post-delivery, and the incidence of neonatal intensive care unit admission. Maternal demographic characteristics such as age, weight, height, gestational weeks, gravida, para, baseline SBP, baseline HR, and EPDS scores at admission were recorded. All primary and secondary outcomes were observed by anaesthesiologists blinded to patient group and dose assignments.The primary outcomes were satisfactory anaesthetic effect until delivery and the incidence of hypotension. Secondary outcomes included maternal hypotension-related symptoms and their management, upper sensory level, total co-load IV fluid volume, blood loss volume, level of sedation during surgery, recovery of lower limb mobility, analgesic effect 24 and 48 h after surgery, and EPDS scores at discharge; neonatal outcomes included induction-delivery interval, umbilical vein blood gas values , and counts and proportions. Parametric data were analysed with the t-test; nonparametric data were analysed with the Mann-Whitney test. Proportions were compared, using the Chi-square and Fisher exact tests, as suitable. The peri-operative haemodynamic parameters were assessed by multiple t-tests and estimated with a two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, with Q = 1%. Statistical comparisons were made, using SPSS for Windows version 18.0  and GraphPad Prism 8 for windows . Statistical significance was defined as The patient recruitment process is presented in Fig. Figures P < 0.001). The dose of rescue phenylephrine bolus administered , and the rates of bradycardia , dyspnoea , nausea , and vomiting  were lower in the RS group than in the R group. The incidence of symptoms associated with S-ketamine such as visceral traction pain  was lower in the RS group than in the R group; in contrast, the rates of dreaming  and dizziness  were higher in the RS group than in the R group. The upper sensory level of spinal anaesthesia was lower in the RS group than in the R group . The levels of sedation during delivery and 5 min after delivery were significantly different between two groups .Maternal outcomes are presented in Table P < 0.001). The recovery of lower limb mobility required less time in the RS group than in the R group . Resting and mobile VAS scores at 24 h  and 48 h  were lower in the RS group than in the R group. The PCA time during 24 h was lower in the RS group than in the R group . The EPDS scores at discharge were lower in the RS group than in the R group .Moreover, the duration of the first PCA was longer in the RS group than in the R group  was different between the groups. The levels of PO2 , BE , and SO2  were higher, and those of lactate  were lower in the RS group than in the R group.Neonatal outcomes are presented in Table P values at 4, 6, 8, and 10 min  were higher in the RS group than in the R group. Although HR values at 2 and 4 min  were higher in the R group than in the RS group, two-stage linear step-up procedures revealed no between-group difference in HR values.Serial changes of SBP and HR values over time after spinal anaesthesia administration are presented in Fig. − 1 may help reduce spinal ropivacaine consumption by 19.7% (95% CI: 19.0 to 26.2%), helping reduce the incidence of hypotension and hypotension-related adverse effects after spinal anaesthesia.In this study, the ED90 of spinal ropivacaine administered with S-ketamine was 11.8 mg (95% CI: 11.7 to 12.7); the corresponding value without S-ketamine was 14.7 mg (95% CI: 14.6 to 16.0), derived with the BCUD method and isotonic regression. The present findings suggest that a single IV S-ketamine bolus of 0.15 mg.kgSeveral methods have been proposed to reduce the incidence or severity of maternal hypotension after spinal anaesthesia during caesarean delivery, including the use intrathecal adjuvant opioids to decrease the total dose of  local anaesthetics used , prophylS-ketamine, the pure dextrorotatory enantiomer of ketamine, is used in clinical analgesia and anaesthesia. The main effects of S-ketamine are mediated by non-competitive inhibition of the N-methyl-D-aspartate receptor; the compound also interacts with opioid receptors, monoamine receptors, and adenosine receptors . It is aFurthermore, it has been demonstrated that a low infusion rate of S-ketamine may reduce opioid consumption and the extent of postpartum delirium , 24. In The faster recovery of maternal lower limb mobility in the RS group was an unexpected finding, likely indicating the contribution of the lower spinal dose of ropivacaine. Satisfactory analgesic effect and faster recovery of lower limb mobility may be direct or indirect results of the ultra-low dose of S-ketamine, corresponding to enhanced recovery after surgery.Anti-depressive effects of ketamine and S-ketamine have been reported in both human and animal studies –27. Howe− 1. Sen et al. [− 1 may produce satisfactory analgesia, while reducing the consumption of post-operative analgesics. Given the 2:1 potency ratio of ketamine and S-ketamine [− 1. We tested this lower dose of S-ketamine in preliminary experiments, failing to induce sleep in most patients; consequently, the dose of 0.15 mg.kg− 1 was used, with only one patient failing to achieve sleep; this patient was sedated in the present study. Moreover, patients undergoing elective caesarean delivery without gastroesophageal reflux or full stomach were included in this study; thus, neither reflux nor aspiration were observed in any of the study patients. Although the coughing, gag, and swallowing reflexes remained functional after the administration of S-ketamine [In the present study, the bolus dose of S-ketamine was 0.15 mg.kgn et al.  and Menkn et al.  previousketamine , the dosketamine , the useThis study has some limitations. First, in this study, both patients and anaesthesiologists were blinded to the spinal dose of ropivacaine administered to each group. However, both the patients and attending anaesthesiologist could identify group assignment based on the hypnotic nature of S-ketamine; however, the resulting biases are unlikely to have influenced the presented findings, as all outcomes were based on separate patient and observer reports. Second, the anti-depressive effect of S-ketamine postpartum was evaluated by the EPDS after post-operative days 4–5, by which point most patients had not recovered either physically or mentally from the impact of caesarean section. Future studies should assess the degree of postpartum depression after ≥6 weeks in this population to comprehensively evaluate the anti-depressive effects of S-ketamine postpartum. Third, the low incidence of maternal hypotension and its related side effects was likely due to the decreased dose of spinal ropivacaine, associated with the use of S-ketamine. Although the effect of stimulating the sympathetic nervous system by S-ketamine has been shown in several previous studies, herein, we could not distinguish between the effect of spinal ropivacaine dose reduction and that of S-ketamine use. Establishing whether an ultra-low bolus dose of S-ketamine increases maternal BP after spinal anaesthesia requires further research. Maternal hypotension tends to occur within 5–10 min after spinal anaesthesia, and if S-ketamine is proved to be able to increase maternal BP, it has the potential to be used earlier in the process than the time point chosen in this study. However, additional evidence is required before this recommendation can be made. Finally, the present study sample was small; large studies are required to validate the safety and side effects of IV S-ketamine for women undergoing elective caesarean delivery.− 1 may be used with caution for patients at high risk of hypotension or experiencing extreme nervousness.The ED90 of spinal ropivacaine was 11.8 mg (95% CI: 11.7 to 12.7) with and 14.7 mg (95% CI 14.6 to 16.0) without S-ketamine. An ultra-low single IV bolus dose of S-ketamine may reduce the required dose of spinal ropivacaine by 19.7% (95% CI: 19.0 to 26.2%), helping reduce the incidence of hypotension and associated symptoms, and helping achieve sedation during caesarean delivery. A spinal ropivacaine of 12 mg with IV S-ketamine bolus of 0.15 mg.kgAdditional file 1.


The U.S. Food and Drug Administration’s (FDA′s) Center for Veterinary Medicine (CVM) has been investigating reports of pets becoming ill after consuming jerky pet treats since 2007. Renal failure accounted for 30% of reported cases. Jerky pet treats contain glycerin, which can be made from vegetable oil or as a byproduct of biodiesel production. Glycidyl esters (GEs) and 3-monochloropropanediol esters (3-MCPDEs) are food contaminants that can form in glycerin during the refining process. 3-MCPDEs and GEs pose food safety concerns, as they can release free 3-MCPD and glycidol in vivo. Evidence from studies in animals shows that 3-MCPDEs are potential toxins with kidneys as their main target. As renal failure accounted for 30% of reported pet illnesses after the consumption of jerky pet treats containing glycerin, there is a need to develop a screening method to detect 3-MCPDEs and GEs in glycerin. We describe the development of an ultra-high-pressure liquid chromatography/quadrupole time-of-flight (UHPLC/Q-TOF) method for screening glycerin for MCPDEs and GEs. Glycerin was extracted and directly analyzed without a solid-phase extraction procedure. An exact mass database, developed in-house, of MCPDEs and GEs formed with common fatty acids was used in the screening. In the late summer of 2007, the FDA became aware of reports of illness in dogs after consuming jerky pet treats (JPTs) ,2. For mJPTs are often made from dried chicken (or duck) meat or sweet potatoes. As of 1 September 2018, FDA’s Veterinary Laboratory Investigation and Response Network (Vet-LIRN) had collected and performed testing on more than 650 JPT samples related to more than 550 consumer complaints, as well as more than 400 retail samples of unopened product bags obtained from a store or shipment. Vet-LIRN did not subject every sample to the entire battery of testing due to limited resources and product availability. Sample testing was targeted based on the concerns with a particular product or brand and the symptoms displayed by the pet that consumed the product. The product-based testing plans targeted the main ingredients  and considered other information on the product labels, such as additional ingredients and/or their contaminants. For example, Vet-LIRN investigation revealed that some products contained glycerin (as high as 20%), although it was not listed on the label, and conducted additional testing for glycerin to follow up on this finding [Glycerin is also used to produce many other products including drugs, cosmetics, foods, tobacco products, sweeteners, and toiletries. As glycerin may be produced during biodiesel production and is subjected to a refining process, it is important to ensure that there are methods to detect potential toxic contaminants that could arise during manufacture.Recently we published a UHPLC/Q-TOF method to screen crude glycerin for toxic phorbol ester contaminants . AnotherFree MCPDs are present in the low mg/kg range in many foodstuffs such as acid-hydrolyzed vegetable protein, soy sauces, crackers, bread, toast and other bakery products, malt, meat products, and soups ,11,12,13An extensive review about 3-MCPDE toxicity was published by the Joint FAO/WHO Expert Committee on Food Additives (JEFCA) in 2016 . In 2018Potential severe toxicological effects induced in animals with the presence of 3-MCPDEs have raised concerns about the level of exposure to these food contaminants. Strong evidence shows that glycidol and GEs are potential genotoxic carcinogen agents and probably carcinogenic to humans . StudiesDue to the potential renal toxicity of MCPDEs, which are derived from MCPD, and the carcinogenicity of glycidol derived from GEs, we focused our method development efforts to screen glycerin for MCPDEs and GEs.Glycerin is the major byproduct of biodiesel production. Centrifugation or gravitational settling is used to separate biodiesel from glycerin after transesterification ,24. The The main factors for the formation of MCPDEs and GEs are the presence of chloride ions; glycerin; and tri-, di-, or monoacylglycerides and high temperatures. As glycerin is subjected to these conditions during production and refining, it is possible that it is tainted with such process contaminants. Many edible oils also undergo a similar refining process to improve their quality. Evidence exists in the scientific literature to show the formation of MCPDEs and GEs during the industrial processing of oils ,25,26,27The determination of MCPDEs is complicated due to structural diversity. Considering the possible positional isomers of MCPD, the formation of about 100 different ester compounds is possible. However, because of the relative abundance of the fatty acids, only seven esters  are considered in food analysis . For GEsLC method development was challenging due to the polarity range of the compounds. We tried to use ACN/water gradients spanning 5–100% ACN in 15 min with formic acid in both solvents. The GEs and the mono-MCPDEs, which are polar, eluted from the column, but the nonpolar di-MCPDEs did not elute from the column with this solvent gradient. We also tried other solvent gradients reported in the literature such as ACN/ MeOH/water as solvent A and acetone as solvent B ramping up to 60% acetone . Even thEven though we used six representative compounds of GEs and MCPDEs in our mixed standard, we analyzed many others in our study. The most polar 1-lauroyl-3-chloropropanediol and the most nonpolar 1,2-distearoyl-3-chloropropanediol eluted within our gradient. The LC retention times ranged from 2.3 to 10.3 min for the six compounds in our mixed standard using this gradient . AverageCalibration curves were generated for selected GEs and mono- and di-MCPDEs in both solvents, methanol and glycerin-matrix . All calThe calibration curves constructed for the mixed standard were used to determine the matrix effect. The calibration standards were used to validate the data analysis process.m/z range of 50–1000 Da and completed within the timespan of the LC gradient. Singly charged ions and a minimum peak height of 10,000 ion counts were selected for FbF. The allowed adduct ions included H+ and Na+. FbF algorithm scores the database matches based on the similarity of each of the isotopic masses (Mass Match), isotope ratios (Abundance Match), isotope spacing (Spacing Match), and optionally, the retention time (RT Match). Data analysis was performed with the Mass Hunter Qualitative Analysis software (B.06.00) using FbF (find by formula) algorithm to screen for compounds in the glycerin extracts using an in-house MCPDE and GE exact mass database as the formula source . The in-FbF algorithm found all six GEs and MCPDEs in the calibration standards, thereby validating the data analysis process see . Our metThe refined and crude glycerin samples we obtained were diluted and subjected to the same screening procedure as the calibration standards. The screen against the database using FbF algorithm produced many false-positive hits for GEs that were eliminated by comparing the retention time and mass accuracy with standards. There were no hits for MCPDEs.The data generated in the study were validated according to the HRMS guidance . The masSamples were analyzed using an Agilent 1290 Infinity UHPLC coupled with 6550 Q-TOF mass spectrometer  in positive-ion mode. Glycidyl linolenate (CAS 51554-07-5), glycidyl linoleate (CAS 243085-63-3), 1-lauroyl-3-chloropropanediol (CAS 20542-96-5), 1-palmitoyl-3-chloropropanediol (CAS 30557-04-1), 2-oleoyl-3-chloropropanediol (CAS 915297-48-2), 1-palmitoyl-2-stearoyl-3-chloropropanediol (CAS 1185060-41-6), 1-oleoyl-2-stearoyl-3-chloropropanediol (CAS 1336935-05-7), and 1,2-distearoyl-3-chloropropanediol (CAS 72468-92-9) were purchased from Toronto Research Chemicals .Sixteen food-grade glycerin samples and four crude-grade glycerin samples were obtained from various sources. A food-grade glycerin sample obtained from Procter & Gamble  was used for constructing matrix calibration curves for the mixed standard.Primary stock solutions (1 mg/mL each) were prepared separately by dissolving the ester standard in methanol. There are two classes of MCPDEs, namely mono- and diesters. Two GEs (glycidyl linoleate and glycidyl linolenate), two MCPD monoesters , and two MCPD diesters  were selected to represent the two classes of MCPDEs and GEs. Working standard solutions were prepared at 50, 100, 500, and 1,000 µg/mL by diluting appropriate volumes of the primary stock solution with MeOH. All solutions were stored in glass vials at 4 °C or below.Appropriate amounts of working standards of MCPDEs and GEs were spiked (added) into methanol and glycerin matrix to produce the following concentrations: 2, 5, 10, 25, 50, 125, 250, and 400 ng/glycerin. Mixed standard in glycerin was prepared using 1 g food grade glycerin (Procter and Gamble) in 2 mL methanol, spiked with appropriate volumes of working standards. Calibration curves were constructed in both methanol and glycerin matrix.Sixteen food-grade glycerin samples and four crude-grade glycerin samples (approximately 1 g each) were prepared separately in 4 mL glass amber vials. Each 1 g glycerin was weighed directly into the vial, and 500 µL of methanol was pipetted into each vial. Vials were capped and vortexed, and methanol was added dropwise to bring the total volume of each sample up to the 2 mL mark. Methanol was used as the solvent blank. Triplicate injections were performed for each sample. We analyzed the diluted spiked glycerin without solid-phase extraction step using the QTOF6500 HRMS mass spectrometer coupled to the UHPLC system. A liquid chromatography system 1290  with a BEHC18 column (2.0 mm × 100 mm) with 1.8 μm particles  at 30 °C was used for HPLC separation. Mobile phase A consisted of 92:8 methanol/water with 0.05% formic acid; mobile phase B consisted of 98:2 isopropanol/water with 0.05% formic acid. The flow rate was 300 µL/min, and the gradient used was as follows: from 0–3 min, mobile phase B 0–30%; from 3–10 min, mobile phase B 30–70%, followed by holding at 70% B for 5 min and returning to 0% B in 2 min. The first 1 min of the LC flow was diverted to the waste to prevent the glycerin matrix from entering the mass spectrometer.m/z < 1700) in the positive ion mode. Data were collected in centroid format. Reference masses at m/z 121.0509 and m/z 922.0089 were continually introduced via a second sprayer for accurate mass calibration. The reference ions used were purine (C5H4N4) at m/z 121.0509 and HP-921 phosphazene (C18H18O6N3P3F24)) at m/z 922.0089 for positive mode. The source conditions were as follows: sheath gas flow, 11 L min−1; sheath gas temperature, 350 °C; nebulizer pressure, 40 psi; drying gas temperature, 150 °C; drying gas flow, 15 L min−1; nozzle voltage, 380 V; fragmentor voltage, 360 V; capillary voltage, 3500 V. Nitrogen was used as source gas and as collision gas. Full scan MS were acquired over the mass range m/z 100–1000 at a scan speed of 2 scans/s. Agilent Mass Hunter workstation software version B.05.00 was used for data acquisition, and B.06.00 qualitative analysis was used for processing. Mass calibration was performed prior to analysis.The Agilent 6550 QTOF instrument  is equipped with an Agilent Jet Stream Technology Dual Spray ESI source and an iFunnel. The instrument was calibrated in the extended dynamic range  and lower mass range . It is a dilute-and-shoot method with minimal sample preparation that can be adapted to other matrices. The screening method by FbF against our in-house library produced many false positives for GEs, which were eliminated by comparing the retention times with standards. There were no hits for MCPDEs in the limited glycerin samples we analyzed.


Renal scintigraphy is widely used to evaluate residual function of a transplanted kidneyfrom the donor. Dynamic computed tomography (CT) imaging can evaluate both kidney morphologyand regional renal function. The aim of this study was to develop an imaging protocol and acalculation method using dynamic CT for assessing the effective renal plasma flow (ERPF) bymodel analysis, and to evaluate the validity of the obtained ERPF values.Preoperative dynamic CT examination with a low radiation dose exposure system wasperformed for 25 renal transplant donors, and ERPF was calculated from the obtained images(CT-ERPF). To calculate CT-ERPF, we set the region of interest (ROI) in the renal cortex usingautomatic ROI-setting software developed in our laboratory. We compared the processing timewith automatic and manual ROI settings. To evaluate the validity of CT-ERPF, we examined thecorrelation of age with CT-ERPF and compared with reported ERPF values. We also compared theuptake rates of technetium-99m-dimercaptosuccinic acid and CT-ERPF in terms of theright-to-left ratio.There was good agreement of CT-ERPF assessed using automatic and manual ROIs.CT-ERPF was negatively correlated with age and showed values below the reference ERPF range in21 cases. The right-to-left ratio of CT-ERPF showed a significant correlation with that oftechnetium-99m-dimercaptosuccinic acid.Using our method, CT-ERPF was a useful indicator for preoperative evaluation ofdonor’s renal function. Renal function can be evaluated preoperatively by several methods,including creatinine clearance, estimated glomerular filtration rate (eGFR), and renalscintigraphy. Morphological evaluation is also performed using dynamic contrast-enhancedcomputed tomography (dynamic-CT) with iodine contrast medium. In our hospital, three-phasedynamic CT is performed for patients with renal disease, and includes pre-contrast of the firstphase, the nephrographic phase, and the excretory phase. Frennby et al.2 This method requires dynamic CT imaging using 20 scansof the first-pass image with one bolus injection of contrast medium. As the area under the curveof the input/output function must be accurately calculated, it is necessary to satisfy thesampling conditions for correct capture of the shape of the time attenuation curve (TDC) at theinterval and the number of dynamic scans. Therefore, if this method is applied as used in320-row multidetector row CT, the exposure radiation dose may be markedly increased. Appropriateminimization of radiation exposure by decreasing the number of scans is important. The purposeof this study was to develop an imaging protocol and calculation method using dynamic CT forcalculation of effective renal plasma flow (ERPF) by model analysis, and to evaluate thevalidity of the obtained ERPF values.A focus of our laboratory is the development of techniques for calculating renalplasma flow from dynamic CT with model analysis. In previous studies, renal blood flow (RBF) wasaccurately calculated using the deconvolution method with dynamic CT imaging.This study was approved by the Health Science Ethics Committee at Fujita HealthUniversity and was conducted in accordance with the Declaration of Helsinki and Good ClinicalPractice. Written informed consent was obtained from all study subjects. Dynamic CT examination with addition of low radiation dose wasperformed for 25 renal transplant donors , and ERPF measurements were performed from the obtained images(CT-ERPF). Contrast medium was injected using a dual-shot GX 7 .Data analysis was performed using MATLAB R2016a  for numericalanalysis programming language and OsiriX version 7.0.4 for medical image management software.c(t)) can be described by Equation 1: k(t) (mg I/g) is the concentration of contrast medium inkidney tissue, and Cg(t) (mg I/g) is the concentration of contrast medium inglomeruli. As time is required for the contrast medium to pass through the glomerulus in theinitial state after contrast administration, the contrast medium appears to accumulate in theglomeruli. Thus, a microsphere model ) is expressed by the amount of contrast medium that continues to accumulatein the renal cortex from the arterial blood, as shown in Equation 2: a(t) (mg I/ml) is the concentration of contrast medium in thearterial blood at time t. From Equation 2, (X(t) and Y(t)) are plotted to obtain an approximatestraight line as X(t)=∫0tCa(τ)dτ, Y(t)=Cc(t). F can be calculated as the slope obtained from the approximate straight line(Patlak plot method).4In a(t) (mg I/ml) is the concentration of contrast medium in thearterial blood of the renal artery. Because the concentration of contrast medium and the CTvalue have a proportional relationship, Ca(t) is represented by the CT attenuationvalue (HU). Pixels in the arterial blood obtained from the CT image have units of HU/voxel,with one voxel represented by one pixel. The contrast medium exists in the plasma, and toobtain Ca(t) from this CT pixel, plasma volume V (ml) of the voxel constituting thispixel must be converted to the contrast medium concentration. This plasma volume is calculatedusing the hematocrit. The renal artery is a large blood vessel corrected for the hematocritvalue of HCTLV in a large blood vessel. The amount of plasma contained in the voxelof one pixel of the renal artery is (1-HCTLV)⋅V (ml), and the concentration of contrast medium contained in the plasma within thisvoxel is Ca(t)(1-HCTLV)⋅V (HU/voxel/ml).To calculate ERPF, hematocrit correction must be performed for renal blood flow Fcalculated from Equation 2. Solving Equation 2 for F yields Equation 3: c(t) is the concentration of contrast medium in the kidney cortex, andrepresents the amount of contrast medium per unit weight. However, the units ofCc(t) obtained from the CT image are CT value per voxel (HU/voxel). First, the unitsof Cc(t) must be converted from CT value per voxel to CT value per unit weight. Whenthe density of renal tissue is assumed to be ρ (g/ml), the weight can be obtained bymultiplying the voxel by the density of the kidney tissue. Therefore, the CT attenuation valueper 1 g of kidney tissue is expressed as (HU·ml/voxel/g).Ca(t). Because the glomerular capillary is a smallblood vessel, the value is corrected for the hematocrit in a small blood vessel,HCTSV. The amount of plasma contained in the voxel represented by one pixel ofkidney cortex is therefore (1-HCTSV)⋅V (ml). The concentration of contrast medium contained in the plasma within thisvoxel is expressed as Ca(t)(1-HCTLV)⋅ρ⋅V (HU/voxel/g).Next, because glomerular capillaries are contained in the kidney cortex, hematocritcorrection is required as for CERPF corrects for the hematocrit in large and small blood vessels, and thecorrection for kidney tissue density is shown in Equation 4: Substituting Equation 3 into Equation 4 yields Equation 5, which converts renalblood flow F to ERPF: LV=0.45 andHCTSV=0.25.5For this study, we performed hematocrit corrections using HCTIn the preoperative examination of donors, CT imaging from the liver to the pelviswas performed, with three phases of pre-contrast, nephrogram, and excretion. For analysis ofERPF using the kinetic model (see previous section), dynamic CT imaging was added before therenal artery phase. The study imaging protocol used dynamic CT with a tube voltage of100 kV, auto exposure control , an X-ray rotationspeed of 0.5 s/rotation, and a 2.0-s interval for 20 s. Imaging was started 8 safter starting injection. Contrast medium was injected at 600 mg I per kg body weightwithin 25 s. The infusion rate was approximately 3.3–5.0 ml/s, with a boostinjection of 30 ml saline. We used FC04 AIDR 3D Strong  for the reconstruction function. The average dose at this time was 29.7 mSv, andthe average dose of dynamic imaging added on this protocol was 4.5 mSv. The contrastmedium used was iopamidol 370 , omnipaque 300 , iomepurol 350 , or Optirei 240 .a(t) . The range from the TDC until the CTvalue started to rise was defined as baseline. An average within the baseline range wascalculated as the baseline value, which was subtracted from the TDC. This corrected TDC wasprocessed with five-point smoothing to average the CT values of five points before and afterthe plot. This value was defined as the input function Ca(t) . The smoIn the phase in which the kidney cortex was being enhanced, three ROIs of thekidney cortex were set in the cross-section of the central part of the kidney, and three TDCswere generated from the obtained ROI. The range from the TDC until the CT value started to risewas defined as the baseline range. CT values of the TDC for each pixel within the baselinerange of the kidney cortex were averaged to determine the baseline value . Determic(t)  .a(t)calculated from the renal artery and output function Cm(t) calculated from the renalmedulla. However, contrast medium in the renal artery flows into the glomerulus and reaches therenal medulla in approximately 1–2 s. Therefore, to accurately perform modelanalysis, the time difference between the input function Ca(t) and renal medullaryoutput function Cm(t) was calculated and corrected.6The methodology assumes no time lag between input function Cc(t). Hematocrit correction was performed on the calculatedvalues for each pixel to prepare an ERPF image. ERPF values of the left and right kidneys, andof both kidneys, were calculated by integrating the ERPF value for each pixel within the ROIset in the renal cortex. For evaluating the accuracy of the automatic ROI of the renal cortex,we compared the processing time for manual and automatic ROIs and the match rate. An ROI of therenal cortex prepared manually and confirmed by radiologists  was evaluated withthe following three items:For creation of an ERPF image and value for one or both kidneys, renal blood flow Fwas calculated with the Patlak method –3 using (1) Match rate between the manual and automatic ROIs. The coincidence rate betweenthe manual and automatic ROIs was calculated using Equation 6: (2) Evaluation of CT-ERPF calculated using manual and automatic ROIs. Thecorrelation of CT-ERPFs calculated using manual ROI with automatic ROI was determined byplotting  the coordinates  to determine a correlation formula, a correlationcoefficient, and a p value.(3) Comparison of processing time between the manual and automatic ROIs. The manualROIs were performed in nine of 25 cases under the guidance of a radiologist employed at FujitaHealth University Hospital. The required time to set the ROI in the renal cortex was comparedbetween manual and automatic settings in nine cases.11 Theconventional paraaminohippuric acid clearance (CPAH) was defined as the referenceERPF. We also evaluated the age-dependence of ERPF values in our cases. To confirm the validityof CT-ERPF values, the right-to-left ratio of technetium-99m-dimercaptosuccinic acid(99mTc-DMSA) kidney uptake and CT-ERPF values before surgery were evaluated.99mTc-DMSA scintigraphy was performed in 19 cases, and was performed first on thesame day of dynamic CT.The calculated ERPF was compared with reported reference values.99mTc-DMSA.Correlation coefficients of those ratios were obtained, and data analyzed by Pearson’scorrelation coefficient. A p-value <0.05 was considered significant. Bland–Altmananalysis was performed to compare differences in the ratios. Bias was determined as the averagevalue of differences in CT-ERPF values calculated from both methods, and limits of agreementwere set from –1.96 to +1.96 standard deviations of the CT-ERPF value.All statistical analyses were performed using statistical software . Statistical comparisons were performed between the CT-ERPF value calculated byautomated ROI and the CT-ERPF value calculated by manual ROI, and between the right-to-leftratio of CT-ERPF values and the right-to-left ratio of renal uptake of The average agreement rate with the manual ROI before manual correction to theautomatic ROI was 81.2%, and the average agreement rate after manual correction was 82.9%. Inboth kidneys, the linear equation was y=1.010×–12.83 (p<0.05), with a linearrelationship close to 0.999 and 1, indicating a significant positive correlation. Thecorrelation of CT-ERPF calculated using the manual and automatic ROIs, and theBland–Altman analysis, are shown in Processing times for setting ROIs in the renal cortex using manual and automaticmethods are shown in PAH) of 442–694 ml/min/1.73 m2,8 whileJapanese studies have reported values of439–817 ml/min/1.73 m2.11 In the present study, all caseswere Japanese adult renal transplant donors . Thus, we adopted a reference range of ERPF (CPAH) of439–817 ml/min/1.73 m2.A representative CT-ERPF image is shown in 99mTc-DMSA kidney uptake with those ofCT-ERPF for 19 of the 25 cases are shown in 99mTc-DMSA uptake and CT-ERPF.Bland–Altman plots had a bias of 0.0087, and the limits of agreement were–0.30–0.32. One case was outside of this range. In this case, the sizes of the left andright kidneys were significantly different compared with other patients.Twenty-one cases showed values below the reference range. The results ofcomparisons of the right-to-left ratio of In our method, the total processing time for automatic ROIs was markedly reducedcompared with that for manual ROIs. Nevertheless, future studies are required to further reducethe total processing time for routine clinical use.2 In this method, dynamic CT imaging requires 20 scansof the first-pass image with one bolus injection of contrast medium. In our proposed modelanalysis method, dynamic imaging is performed in the initial phase of contrast medium flowinginto the cortex, and the input function from the renal artery image and the cortical area areused to calculate the output function for each pixel TDC. A time difference appears in theimage because of differences in the flow path of the contrast medium. As the model usescalculations assuming no time difference between the paths, difference correction isnecessary.8 According to Krieret al.,12 output function of thecortex appears as the initial vascular phase, then in the proximal tubule, followed by thedistal tubule. To accurately capture the shape of the vascular phase, the latter two phasesmust be separated in the deconvolution method. Thus, at least two phase TDCs of the vascularand proximal tubules are required. Compared with the deconvolution method, the Patlak methodcalculates RBF using several points of the initial vascular phase of TDC. Thus, the number ofdynamic scans (sampling number) can be reduced.RBF can be accurately calculated by the deconvolution method using TDC.5 Thus, in the present study the concentration ofcontrast medium in the renal cortex was converted from the CT value per voxel to the amount ofcontrast medium per unit weight,6 and thedensity of renal tissue was the same as that of Kudo et al.5 To obtain the ERPF, we used hematocrit correction of large and smallblood vessels, and used Equation 7 to consider the density of kidney tissue.Kudo et al. performed hematocrit correction with HCTLV=0.45, HCTSV=0.25, and abrain tissue density of 1.04 g/ml to measure cerebral blood flow.PAH; clearance ofiodine-131-ortho-iodohippurate (COIH)=0.90 CPAH; clearance oftechnetium-99m-mercaptoacetyltriglycine (99mTc-MAG3) (CMAG3)=0.59; andCOIH=0.53 CPAH.8 We considered that CT-ERPF was not equal toCPAH, and that CT-ERPF could be converted by the additional factor. CT-ERPF valuesdecreased with age. Further, Russell et al. reported a decrease in ERPF with aging, whichwas calculated using the following equation for patients >40 years: ERPF=568–5.83 (age–40)±126.8 Dujardin et al.13 alsoreported an inverse correlation of age with RBF calculated from contrast MRI data using thedeconvolution method. This was because of a moderate age dependence of these values in males.Further, arterial blood flow from Doppler ultrasound measurements tended to decrease withage.15 We also found a trend towards a decrease in CT-ERPF with age, which mayreflect a progressive decrease in renal function.CT-ERPF values using the Patlak method in our study tended to be lower (21 of 25cases) than reported reference ERPFs. Russell et al. used these measurementsinterchangeably with the following conversions: ERPF=C99mTc-DMSA uptake. Taylor reported an excellent agreement of99mTc-DMSA uptake and COIH with serum creatinine≤2.0 mg/dl,16 while Mominet al. reportedthat 99mTc-DMSA and technetium-99m-diethylenetriaminepentaacetic acid(99mTc-DTPA) scanning methods provided similar relative renal functionsvalues.17 Thus, CT-ERPF values may reflecta relative renal function similar to the GFR in the donor’s kidney.In the present study, there was an excellent correlation of the right-left ratio ofCT-ERPF with that of PAH) wasdifficult in routine examination in our hospital. For this reason, we performed indirectcomparisons and evaluations of the distribution of normal values and nuclear medicineexamination findings. Historically, examination by 99mTc-MAG3 is common for renalblood flow examination. However, in our university hospital, the 99mTc-DMSA kidneyuptake test was routinely performed for split renal function. Another limitation is that GFR should be used toevaluate renal function. Our present data are preliminary findings, and simultaneousmeasurements of CT-ERPF and CT-GFR are in progress in our laboratory using the same method.These measurements may be useful for assessing kidney diseases. Finally, the dose of radiationexposure on CT was higher compared with radio-isotope examinations, for example,99mTc-DTPA and 99mTc-MAG3.18 However, we suggest that CT provides detailed anatomical information beforetransplantation surgery compared with other examinations. Thus, in limited cases, such as inrenal transplantation donors, CT is a useful technique and should be used.A limitation of our study is that measurement of RBF (e.g., CDynamic CT enables calculation of CT-ERPF using our proposed method and is afeasible technique in renal transplantation donors.


One-year old children were randomised to receive a reduced-protein GUMLi (1.7 g protein/100 mL) or a non-fortified CM (3.1 g protein/100 mL) for 12 months. Blood sampling and anthropometric measurements were made at 1 and 2 y. Diet was assessed using a validated food frequency questionnaire. Total protein intake (g/d) from all cow's milk sources was 4.6 g  lower in the GUMLi group after 12 months of the intervention, with a significant group-by-time interaction (p = 0.005). Length-for-age (LAZ) and weight-for-length (WLZ) z-scores did not differ between groups, however, mean body fat % (BF%) was 3.2%  lower in the GUMLi group at 2 y. There was no difference between the intervention groups in relation to IGF-1 and IGF-BP3 , with no group-by-sex interaction. After combining the groups, IGF-1 concentration at 2 y was positively correlated with parameters of growth , total cow's milk intake (p = 0.032) after adjusting for sex, breastfeeding status, and gestation. Randomisation to a reduced protein GUMLi resulted in small reduction in %BF and lower total protein intakes but had no effect on growth. Plasma IGF-1 concentrations were independently associated with total protein intake from cow's milk at 2 y, highlighting a potential area of the diet to target when designing future protein-related nutrition interventions.The relationship of protein intake with insulin-like growth factor 1 (IGF-1) concentrations in well-nourished children during the second year of life is poorly understood. The aim of this study was to explore the effect of a reduced-protein Growing Up Milk Lite (GUMLi) or unfortified cow's milk (CM) on protein intake, growth, and plasma IGF-1 at 2 y. An exploratory analysis of a sub-sample of Auckland-based children (Clinical Trial Registration: Australian New Zealand Clinical Trials Registry number: ACTRN12614000918628. Date registered: 27/08/2014. During the transition to a family-style diet, a child's protein intake increases significantly, often exceeding physiological demands and creating an imbalance between age-related protein requirements and growth velocity , 2. The z-score during the first 2 y of life and beyond has been reported  or Young Child Formula (YCF), which are often a significant protein source in a child's diet , 5, 25 aThis study represents an exploratory analysis of the GUMLi Trial in a sub-sample of Auckland-based children. The GUMLi trial was a multi-centre double-blind randomised, comparator-controlled trial designed to compare the effect on body composition at 2 y of a reduced protein GUM (GUMLi) fortified with iron, vitamin D, pre- and probiotics (synbiotics) vs. unfortified CM as part of a whole diet for 12 months . The trin = 108) and Brisbane (n = 52). Participants were randomised 1:1 to one of two treatment groups: GUMLi or whole pasteurised and homogenised, unfortified cow's milk (CM), both provided in powder form for a period of 12 months. Surplus plasma samples were available for the current analysis in a sub-sample of Auckland children only (n = 79) at baseline and month 12 of the intervention. No surplus samples were available from children enrolled in the Brisbane arm of the GUMLi trial.Healthy children, 1-year of age (±2 weeks) were recruited from Auckland (The full study design is described in detail elsewhere . BrieflyDietary intake was assessed using the interviewer-administered Eating Assessment in Toddlers Food Frequency Questionnaire (EAT FFQ) at baseline and month 12 of the intervention , 31. The2), and body composition determined by a single frequency bioelectrical impedance (BIA) device (Bodystat 1500 MD) were performed by trained GUMLi study staff and have been described in detail elsewhere (Standardised anthropometric measurements including weight (kg), recumbent length (cm), BMI (kg/mlsewhere .A non-fasted 3 mL finger prick capillary sample was collected by an experienced paediatric phlebotomist at baseline and month 12 of the intervention. Samples were collected into a 0.25 mL EDTA tube (full blood count), two 0.5 mL serum separate tubes (c-reactive protein and iron studies) and a plain 0.5 mL tube for 25-hydroxyvitamin D concentration measurements. Within 2 h of collection, blood in the serum separator tubes was centrifuged for 10 min at 3,000 rpm to separate the serum from whole blood. Samples were analysed by the local pathology laboratory, LabTests Auckland (c-reactive protein and iron studies) as part of the trial secondary analyses . LabTestp > 0.05 for either test treated as normally distributed. All model assumptions were checked and statistical tests were two-sided with a significance level of p < 0.05. Baseline variables between the GUMLi and CM groups were compared using independent t-tests for parametric variables and Mann-Whitney for non-parametric variables. For categorical variables, chi-squared tests were used, or Fisher's exact test with small cell counts (<5).Baseline descriptive statistics, including participant and parental characteristics were summarised by treatment group. Categorical variables were described as frequencies and percentages, normally distributed continuous variables as means ± SD, and non-normal continuous data as medians and quartiles. IGF-1 and IGFBP-3 concentrations were not normally distributed and were log transformed and the geometric mean displayed. All statistical analyses were performed using SPSS, version 26 for Windows . Data was tested for normality using Kolmogoroc-Smirnov and Shapiro-Wilk tests, with t-test was used as a post-hoc analysis to compare values between groups at each time point (paired) and over time (independent) and changes over time within each group (paired). ANCOVA regression models were used to investigate the effect of the study milks on anthropometric, dietary and biochemical variables after 12 months of the intervention, adjusting for baseline outcomes, and child sex. A hierarchical multiple linear regression was used to test associations between IGF-1 and variables (growth and nutrient intake), adjusting for confounders. Partial correlations were performed on the sub-sample to identify relationships between growth, biochemical and dietary intake variables, controlling for sex, whether the child was still breastfeeding at 2 y, and gestation due to their known relationships with IGF-1.Repeated-measures ANOVA were used to evaluate the main effect of time and group and their interactions with the dependent variables. Student's n = 108) had biochemical samples available for IGF-1 and IGFBP-3 concentration analysis at both baseline and month 12 of the intervention (n = 6) of the CM children were reported as being born <37 weeks gestation compared to 0% in the GUMLi intervention (p = 0.019). Three of the children born <37 weeks and randomised to the CM intervention were VLBW (n = 1) or low birth-weight  and 2 children randomised to the GUMLi intervention were LBW. The remaining child with a gestation of <37 weeks was not considered low birth weight. Forty percent of the participants were receiving breastmilk at baseline. There were no group differences in anthropometric characteristics at baseline, and prior to randomisation total baseline energy and protein intakes did not differ between groups.A total of 79 (73%) children from the Auckland centre of the GUMLi trial (full Auckland cohort rvention . Baselinp = 0.423). Energy-adjusted daily protein intake was 2.8 g/1,000 kcal  lower in the GUMLI group after adjusting for baseline protein intake and child sex. Total protein intake (g/d) from all cow's milk drink sources was 4.6 g  lower in the GUMLi group after 12 months of the intervention. Notably, percent total protein intake (%PI) from cow's milk was 6.9%  lower in the GUMLi group at month 12. This resulted in a 4% increase and 30% decrease in %PI from cow's milk at month 12 in the CM and GUMLi groups, respectively.Total energy  and absolute protein intakes (g/d) increased over time, however, they did not differ significantly between groups at baseline or month 12 of the intervention . There wz-scores and measures of adiposity are summarised in p = 0.032) lower body fat percentage (BF%) at 2 y compared to children randomised to receive CM, however, no significant group-by-time interaction was seen.Mean weight, length, BMI measurements, their associated p = 0.0.073) lower than children in the CM group. IGF-1 significantly increased from 1 to 2 years of age (p = 0.003) in both groups, however, IGFBP-3 did not change significantly between 1 and 2 years of age and were comparable between groups . There was a significant positive correlation between IGF-1 and protein per gramme of body weight (p = 0.020) and %EI from cow's milk protein (p = 0.044). After 12 months of the intervention, IGF-1 at 2 y remained positively correlated with size  after adjustment for sex, breastfeeding status, and gestation. Change in IGF-1 from 12 to 24-months-of-age was significantly positively correlated with weight . There was a significant association between IGF-1 and total cow's milk intake  and a trend towards an association between %EI from cow's milk protein  adjusted for sex, breastfeeding status, and gestation (There was no effect of the milk intervention on IGF-1 concentrations at month 12 of the intervention . Therefoestation .p = 0.03).Further, to establish whether associations between IGF-1 at 2 y and protein intake from cow's milk were independent of confounding, a hierarchical multiple linear regression was performed incorporating sex and LAZ at 2 y in the base model . LAZ wasIn this sub-set of well-nourished, healthy, Auckland-based children from the GUMLi randomised control trial cohort we found no effect of the milk interventions on IGF-1 concentrations at 2 y across the two groups or by sex. As expected, adjusted protein intake , total protein intake from cow's milk-based drinks, and %EI from cow's milk protein decreased significantly in the GUMLi group over the 12-month duration of the trial, with significant group-by-time interactions evident at 2 y. There were no independent effects of the milk intervention at 2 y for anthropometric variables, except BF% and FFM.Post-hoc analysis revealed a significant decrease in WLZ over the 12-month duration of the study in the GUMLi group only, suggestive of a decreased risk of overweight in the GUMLi group, further supported by the small but significant difference in BF% between groups alongside significant linear growth. These growth patterns are similar to those observed in children randomised into a dairy vs. a meat-based complementary diet from 5 to 12 months of age reported by Tang et al.  at 2 y was independently associated with IGF-1 concentrations at 2 y. The cross-sectional relationship between energy-adjusted protein intake and IGF-1 concentrations in healthy, well-nourished children at 2 y is similar to that reported by Larnkjaer et al. , where IA limitation of this analysis, is that it is an exploratory analysis of the GUMLi randomised controlled trial which was originally powered to detect a 0.5 SD of difference in body fat percent at 2 y and not differences in IGF-1 concentrations or other anthropometric measurements. Excess frozen serum samples were only available for a subset of the Auckland cohort and not the Brisbane cohort. However, the Auckland cohort encompassed 68% of the GUMLi trial sample. Serum urea nitrogen (SUN), an indicator of total protein intake was not measured due to limited serum sample volume and would have strengthened this analysis as increases in SUN have been reported in 9 and 12-month-old infants randomised to a whole milk intervention, with a trend for difference between milk intervention groups of whole milk and formula . AbsolutWhilst the reduced protein GUMLi milk intervention in comparison to CM resulted in a small but significant effect on percentage body fat, it had no effect on IGF-1 concentrations at 2 y. Protein intake from CM was positively associated with IGF-1 concentrations on cross-sectional analysis at 2 y. CM remains an important source of nutrition in young children, providing nutrients outside of protein, however, further consideration as to how much it contributes to total protein intakes in early childhood is warranted. The second year of life should be considered as a specific time point where protein-specific dietary interventions could be targeted as children transition to the family diet in an effort to reduce the risk of overweight and adiposity in later childhood.The raw data supporting the conclusions of this article will be made available by the authors upon request.The studies involving human participants were reviewed and approved the Northern B Health and Disability Ethics Committee (HDEC) of the New Zealand Ministry of Health (HDEC reference number 14/NTB/152) and the University of Queensland Medical Research Ethics Committee (MREC) in Brisbane, Australia (reference number 2014001318). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.CW, PD, and CG: developed the GUMLi Trial. AL, MM, TM, and RH: conducted the study data. AL and CW: wrote the manuscript. AL: conducted the statistical analyses of the data. All authors have read, contributed, and approved the final manuscript.AL has received honoraria for presentations and consultations from Danone Nutricia. CW has received honoraria for presentations and consultations from Danone, Nutricia, Pfizer, and Fonterra. CG has received honoraria for consultations from Fonterra. PD has received honoraria for presentations and consultation from Danone Nutricia, Nestle, Bayer, Diary Australia, H+H, Bellamys, Sanulac, and Aspen Nutritionals. RH is currently employed by Reckitt Benckiser Group, however was employed at the University of Queensland at the time of the GUMLi Trial. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.